Quantitative analyte assay device and method

Abstract

The present invention relates to an assay device and a method for using such for the quantitative determination of an analyte, based on a test strip, which contains a porous test membrane allowing for capillary flow of the analyte and complexes of the analyte, a porous upstream membrane in fluid connection with the test membrane and a porous downstream membrane in fluid connection with the test membrane, wherein the test membrane contains a test site having immobilized thereon a ligand capable of reacting with the analyte and binding such to the test site, and two standard band sites having immobilized thereon known high and low concentrations of a calibrator agent capable of reacting with a label conjugate and binding such to the standard sites, wherein the upstream membrane has a site for the application of a sample to be analyzed, and has a site downstream from the sample application site for depositing label conjugates capable of reacting with the analyte and label conjugates capable of reacting with the immobilized calibrator agents in the standard bands to provide a known label response in the standards bands, and the downstream membrane is capable of absorbing said sample and providing the capillary flow for the sample through the upstream and test membrane.

What is claimed is: 
     
         1 . An assay device for the quantitative determination of an analyte, comprising a test strip containing a porous test membrane allowing for capillary flow of the analyte and complexes of the analyte, a porous upstream membrane in fluid connection with the test membrane and a porous downstream membrane in fluid connection with the test membrane, (a) said test membrane containing a test site having immobilized thereon a ligand capable of reacting with the analyte and binding such to the test site, and at least two standard band sites having immobilized thereon known high and low concentrations of at least two different calibrator agents capable of reacting with a label conjugated antibody and binding such to the standard sites, (b) said upstream membrane having a site for the application of a sample to be analyzed, and having a site downstream from the sample application site for removably depositing label conjugates capable of reacting with the analyte and at least two label conjugates capable of reacting with the immobilized calibrator agents in the standard bands to provide a known label response in the standards bands, and (c) said downstream membrane capable of absorbing said sample and providing the capillary flow for the sample through the upstream and test membrane. 
     
     
         2 . The assay device of  claim 1 , wherein the label conjugates on the upstream membrane give rise to a color. 
     
     
         3 . The label conjugates of  claim 2 , comprising gold conjugates, fluorescent dye conjugates, latex conjugates and enzyme conjugates. 
     
     
         4 . The device of  claim 1 , wherein the test membrane contains two calibrator agents. 
     
     
         5 . The device of  claim 4 , wherein the calibrator agents are rabbit IgG and bovine thyroid globulin. 
     
     
         6 . The device of  claim 1 , wherein the test strip is contained in a cassette allowing for the addition of the sample at the upstream membrane and viewing the results of color formation at the sites of the test membrane. 
     
     
         7 . The device of  claim 1 , wherein the label conjugates are different for the analyte and the calibrator agents. 
     
     
         8 . The device of  claim 1 , wherein the test membrane is microporous nitrocellulose and the upstream membrane is fiberglass. 
     
     
         9 . The device of  claim 1 , wherein the test membrane contains three standard bands. 
     
     
         10 . The method of  claim 7 , wherein the label generates a visual signal at the binding sites of the assay device. 
     
     
         11 . The method of  claim 10 , wherein the visual signal is generated by a colloidal gold conjugate, a fluorescent dye conjugate, or a latex particle. 
     
     
         12 . The method of  claim 1 , wherein the analyte is an antigen associated with an infectious agent such as a virus, bacterium fungus or a prion. 
     
     
         13 . The method of  claim 1 , wherein the analyte is a hormone such as hCG, thyroxin, TSH, glucagons, insulin relaxin, prolactin, melanotropin, gastrin, somatotropin, gastrin and bradykin. 
     
     
         14 . The method of  claim 1 , wherein the analyte is a cancer or tumor marker such as prostrate specific antigen (PSA), carcinoembryonic antigen (CEA), and alpha-fetoprotein. 
     
     
         15 . The method of  claim 1 , wherein the analyte is a cardiac marker. 
     
     
         16 . The method of  claim 1 , wherein the calibrator agents are monoclonal ligands. 
     
     
         17 . The method of  claim 16 , wherein the calibrator agents are BTG antibodies, rabbit IgG antibodies and the label conjugates are label bound antigens capable of binding to the calibrator agents. 
     
     
         18 . The method for the quantitative analysis of an analyte in a sample comprising (a) immobilizing on a microporous test strip in a test band an antibody to the analyte to be established in concentrations adequate to bind to analyte concentrations in the sample, (b) immobilizing different calibrator agents on the test strip in at least two standard bands in the amounts necessary which when treated with excess of labeled antibodies to the immobilized calibrator agent result in a measurable response corresponding to different concentration of analyte, (c) removably depositing on an additional upstream membrane in contact with the test strip labeled antibodies to the analyte and the calibrator agents (d) establishing the curve showing the measurable conjugate response to different analyte concentration, (e) adding a sample suspected of containing the analyte for which the test strip was designed to the assay strip and complexing such to an antibody conjugated to a measurable label and adding measurable labeled antibodies to the calibrator agents to the sample prior to contact with the standard and test bands, (f) measuring the label response at the test and standard bands and comparing such to the standard curve of step (d) for the label responses in the standard bands as established in step (b) and (g) converting the measured label responses to concentration of analyte in the sample.